Novel Gonadotropin-Releasing Hormone, Precursor Peptides Thereof and Genes Encoding the Same

ABSTRACT

A novel peptide found as gonadotropin-releasing hormone (GnRH) in octopuses, a member of mollusks. The structure and the activities of the peptide have been unraveled. The peptide finds use as a reagent used in the studies of correlations between the structure and the activities of GnRH and the information processing mechanisms of the nervous systems in higher animals. It can also serve as a base compound in the development of pesticides and drugs. The peptide has the GnRH activity and has the following amino acid sequence (I): 
                     (I)                   &lt;Glu-Zaa-Zaa-His-Zaa-Ser-Zaa-Zaa-Zaa-Zaa-Pro-               Gly-NH 2                         
wherein &lt;Glu represents pyroglutamic acid and Zaa represents any amino acid. Specifically, the peptide has the following amino acid sequence (1):
 
                     (1)                   &lt;Glu-Asn-Tyr-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-               NH 2                         
wherein &lt;Glu represents pyroglutamic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 10/312,637 filed Dec. 30,2002 (now U.S. Pat. No. ______), which was a 371 of International PatentApplication No. PCT/JP01/05607 filed Jun. 29, 2001, the entire contentsof both of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a novel neuropeptide and, moreparticularly, to a novel peptide that is obtained from brains of Octopusvulgaris (including optic glands, optic tracts, and optic lobes) and hasthe gonadotropin-releasing hormone (GnRH) activity. The presentinvention also relates to a precursor polypeptide of such a peptide anda gene encoding the same.

BACKGROUND ART

Invertebrates have a complex endocrine system that has developed alongwith their nervous system. In invertebrates, various endogenous peptidesare known that serve as neurotransmitters, neuromodulators orneurohormones.

Mollusks generally have a simpler nervous system as compared to higheranimals. Their nervous systems therefore serve as a useful tool instudying the mechanisms of information processing. Any knowledgeobtained at cellular level about the information processing mechanismsof mollusks is considered generally applicable to the informationprocessing mechanisms of nervous systems of higher animals. For thisreason, a significant amount of effort has been made to findneurotransmitters of mollusks.

For example, an endogenous neuropeptide obtained from mussel isdisclosed in Japanese Patent Laid-Open Publication No. Hei 1-221392. Anovel peptide isolated from ganglia of Achatina fulica is disclosed inJapanese Patent Laid-Open Publication No. Hei 2-286696. This peptideincludes a D-amino acid and is found to function as an endogenousneuro-transmitter. Japanese Patent Laid-Open Publication No. 6-56890discloses a neuropeptide isolated from ganglia of a Hungarian snail.

Aside from those described above, various other neuropeptides have beenisolated from mollusks and have been identified. Examples includeAla-Pro-Gly-Trp-NH₂ (SEQ ID NO: 17), myomodulin-CARP, small cardioactivepeptide (SCP), buccalin, and Phe-Met-Arg-Phe-NH₂ (SEQ ID NO: 18)(FMRF-amide) (M. Kobayashi and Y. Muneoka, Zool. Sci., 7, 801 (1990); Y.Muneoka and M. Kobayashi, Experimentia 48, 448 (1992); Youjiro. Muneoka,Journal of Pesticide Science, 18, S191 (1993); Y. Muneoka, T. Takahashi,M. Kobayashi, “Perspective in Comparative Endocrinology”, NationalResearch Council of Canada, 1994, p109; A. Di Cosmo and C. Di Cristo, J.Comp. Neurol., 398, 1-12 (1998)).

Mollusks form one of the largest families of invertebrates. Of differentspecies of mollusks, octopuses belong to a group called Cephalopoda. Theanimals have highly developed brains and far more advanced motor andsensory functions than other members of mollusks. The neurosecretorysystem of octopuses has been extensively studied. In 1956, Boycott andYoung discovered that cutting the optic tract of the animal results inenlarged optic glands and development of gonads. Later, the Wells showedthat the gland secretes a gonadotropic hormone (M. J. Wells and J.Wells, J. Exp. Biol., 36, 1-33 (1959)). It turned out that the hormoneis a protein hormone and affects the growth of germ cells, thematuration of gonad-related organs, and the synthesis of yolk protein.The hormone is also involved in parenting behaviors of octopuses aftertheir laying eggs (Atlas of endocrine organs, Japan Society forComparative Endocrinology. ed., Kodansha).

Gonadotropin-releasing hormone (GnRH) was first isolated in 1971 bySchally et al., from the porcine hypothalamus and was then discovered asluteinizing hormone-releasing hormone (LHRH). Its structure was alsodetermined. Since then GnRHs have been studied by many researchers. Todate, many GnRHs have been found in different vertebrates such asmammals, birds, and salmon. Also, some suggest that many invertebratesinclude tissues and substances that are immunoreactive to GnRH (A. DiCosmo and C. Di Cristo, J. Comp. Neurol., 398, 1-12 (1998)).

It has been shown that, in mammals, GnRHs are secreted fromhypo-thalamus and act on the pituitary gland to cause the release ofgonadotropic hormones. These GnRHs are each a decapeptide.

In octopuses, however, the structure of the gonadotropic hormone has yetto be determined although it is known, as described above, that thegonadotropins are secreted from their optic glands. Moreover, no GnRHhas yet been isolated from octopuses, nor has its structure beendetermined.

Accordingly, it is an objective of the present invention to identify,and determine the structure of, a novel GnRH of octopuses. It is anotherobjective of the present invention to understand the activities of theGnRH, the structure of which is determined according to the presentinvention, both in octopuses and in other invertebrates and vertebrates.

A further objective of the present invention is to provide a novelpeptide that can serve as a useful reagent in studying correlationsbetween the structure and the activities of GnRH and in studying theinformation processing mechanisms of nervous systems in higher animals.A still further objective of the present invention is to provide a novelpeptide that can be used as a base compound in the development ofpesticides and drugs.

It is yet another objective of the present invention to identify aprecursor polypeptide of the GnRH and a gene encoding the precursor forthe purpose of expanding our knowledge about evolution of animals. It isyet another objective of the present invention to provide a method forproducing the GNRH.

DISCLOSURE OF THE INVENTION

In an effort to isolate GnRH from octopuses, the present inventorsconducted extensive studies on various physiologically active peptidesobtained from the brains of about 100 specimens of O. vulgaris byinvestigating the activities of the peptides to enhance the heart beatof the animals. As a result, the present inventors isolated and purifieda peptide having the following amino acid sequence (1), determined itsstructure, and verified the structure by total synthesis:

(1) (SEQ ID NO: 1) <Glu-Asn-Tyr-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly- NH₂wherein <Glu represents pyroglutamic acid. This peptide may be referredto as Compound (1) hereinafter. Unless specified, all amino acids arerepresented by the three-letter codes defined by the IUPAC nomenclaturesystem.

As shown in FIG. 5, Compound (1) has a very high homology to the GnRHsidentified in vertebrates. Unlike known decapeptide GnRHs, Compound (1)turned out to be a dodecapeptide.

The present inventors further conducted tests using quails and provedthat Compound (1) has the activity of GnRH, namely, the activity tocause the release of luteinizing hormone (LH) from the pituitary glandof quails.

The present inventors also prepared a primer from the above amino acidsequence and performed a gene sequence analysis using the reversetranscriptase polymerase chain reaction (RT-PCR) on the total RNAobtained from the brains of O. vulgaris. In this manner, the presentinventors determined that the precursor polypeptide of the peptide ofthe present invention has a total primary amino acid sequence shown asSEQ ID NO: 2.

The present inventors further determined that the gene encoding theprecursor polypeptide has a base sequence shown as SEQ ID NO: 3, therebycompleting the present invention.

Moreover, taking advantage of the decapeptide GnRH identified invertebrates, the present inventors synthesized a new peptide similar tothe peptide of the amino acid sequence (1) isolated from the brains ofO. vulgaris by inserting two amino acid residues, Asn-Tyr, into theN-terminal region of the decapeptide GnRH between the first <Glu and thesecond His residues and examined its physiological activities.

Among other GnRHs, chicken-II-type GnRH (cGnRH-II) shown in FIG. 5 iscommonly found in various classes of animals as well as in chickens. Forthis reason, the two amino acid residues, Asn-Tyr, were inserted intothe N-terminal region of cGnRH-II between the first <Glu and the secondHis residues of the peptide to give a new peptide represented by thefollowing amino acid sequence (2):

(2) (SEQ ID NO: 5) <Glu-Asn-Tyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- NH₂wherein <Glu represents pyroglutamic acid. The peptide may be referredto as Compound (2) hereinafter. Compound (2) was examined for theactivity to enhance the heart beat of O. vulgaris and it was observedthat Compound (2) showed an activity similar to Compound (1) isolatedfrom the brains of O. vulgaris whereas cGnRH-II showed no such activity.

Furthermore, the present inventors deleted amino acids 2 and 3 from theN-terminal region of the dodecapeptide, which was isolated frominvertebrates and had the GnRH activity, to convert it into adecapeptide similar to the GnRH isolated from vertebrates. As a result,it was observed that the decapeptide retained the initial GnRH activity.

To rephrase, the present inventors confirmed that the peptiderepresented by the following amino acid sequence (3), which is obtainedby deletion of amino acids 2 and 3 from the N-terminal region ofCompound (1) isolated from the brains of O. vulgaris, exhibits the GnRHactivity:

(3) (SEQ ID NO: 6) <Glu-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH₂wherein <Glu represents pyroglutamic acid. The peptide may be referredto as Compound (3) hereinafter.

Accordingly, the present invention provides a dodecapeptide having theactivity of gonadotropin-releasing hormone (GnRH) and having thefollowing amino acid sequence (I):

(I) (SEQ ID NO: 7) <Glu-Zaa-Zaa-His-Zaa-Ser-Zaa-Zaa-Zaa-Zaa-Pro-Gly- NH₂wherein <Glu represents pyroglutamic acid and Zaa represents any aminoacid.

The amino acid sequence (I) of the above-described peptide having theGnRH activity may be more specifically defined by the following aminoacid sequence (II):

(II) (SEQ ID NO: 8) <Glu-Zaa-Zaa-His-Zaa-Ser-Zaa-Gly-Zaa-Zaa-Pro-Gly-NH₂wherein <Glu represents pyroglutamic acid and Zaa represents any aminoacid.

The amino acid sequence (I) of the above-described peptide having theGnRH activity may be even more specifically defined by the followingamino acid sequence (III):

(III) (SEQ ID NO: 9) <Glu-Zaa-Zaa-His-Yaa-Ser-Zaa-Gly-Zaa-Zaa-Pro-Gly-NH₂wherein <Glu represents pyroglutamic acid, Zaa represents any aminoacid, and Yaa represents aromatic amino acid.

Of the peptides having the GnRH activity provided by the presentinvention, particularly preferred are those represented by the followingamino acid sequences (1), (2) and (3):

(1) (SEQ ID NO: 1) <Glu-Asn-Tyr-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly- NH₂(2) (SEQ ID NO: 5) <Glu-Asn-Tyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- NH₂(3) (SEQ ID NO: 6) <Glu-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH₂wherein <Glu represents pyroglutamic acid.

The present invention further provides a method for producing apharmaceutical agent or a pesticide containing as an active ingredientthe peptide of the amino acid sequence (I) having the GnRH activity, aswell as a method for producing juvenile octopuses.

According to one aspect of the present invention, there is provided aprecursor polypeptide having either an amino acid sequence of SEQ ID NO:2, an amino acid sequence obtained by deletion or substitution of partof the amino acid of SEQ ID NO: 2, or an amino acid sequence obtained byaddition of one or more amino acids to the amino acid sequence obtainedby deletion or substitution of part of the amino acid of SEQ ID NO: 2.

According to another aspect of the present invention, there are provideda gene encoding the peptide and the precursor polypeptide of the presentinvention, in particular a gene having a base sequence of SEQ ID NO: 3,a vector incorporating these genes, and a host transformed by using thevector, and a method for producing, by means of genetic engineering, thepeptide or the precursor polypeptide of the present invention by takingadvantage of these genes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting the results of reversed-phase HPLC showingthe final elution pattern of Compound (1), a peptide according to thepresent invention in Example 1.

FIG. 2 is a diagram showing the heart beat of O. vulgaris when an amountof Compound (1), the peptide of the present invention, corresponding toone specimen of O. vulgaris was added to a chamber in Example 5.

FIG. 3 includes diagrams showing the results of Example 5, wherein thediagrams show variation in the heart beat of O. vulgaris (a) when 10⁻⁵ McGnRH-II was added to the chamber and (b) when 10⁻⁵ M Compound (2), thepeptide of the present invention, was added to the chamber,respectively.

FIG. 4 is a graph showing the amounts of released luteinizing hormone(LH) when Compound (1), the peptide of the present invention, wasapplied to the pituitary gland of a quail in Example 6.

FIG. 5 is a comparative diagram showing structures of Compound (1), thepeptide of the present invention, and of gonadotropin-releasing hormonesidentified in different vertebrates. The figure discloses SEQ ID NOS:19-26 and 1, respectively, in order of appearance.

FIG. 6 shows the amino acid sequence of the precursor polypeptide of thepresent invention, in which the part corresponding to the peptide of thepresent invention is underlined. The disclosed nucleotide sequence isSEQ ID NO: 3.

BEST MODE FOR CARRYING OUT THE INVENTION

Novel peptides provided by the present invention act to cause therelease of luteinizing hormone (LH) from the pituitary gland in quailsand to enhance the heart beat in Octopus vulgaris. For this reason, thepeptides can be isolated and purified from the brains of O. vulgaris onthe basis of this activity toward the hearts of O. vulgaris as follows:

In one example, hot water extracts of brains of O. vulgaris areprepared. To the extract solution, acetic acid is added to aconcentration of 3%. The solution is allowed to cool and is thencentrifuged to obtain crude extracts. The extracts are adsorbed onto aC18 cartridge (e.g., Sep-Pak® Vac C18 cartridge manufactured by WATERSCo., Ltd.) and are then eluted with a 60% methanol containing 0.1%trifluoroacetic acid to collect a peptide fraction. The peptide fractioncan be subjected to ion-exchange chromatography, reversed-phasechromatography, or other chromatography techniques to separate andpurify peptides of interest.

Each being a dodecapeptide, the peptides of the present invention can bereadily synthesized by solid-phase synthesis using a common peptidesynthesizer (e.g., Peptide synthesizer 433A, manufactured by PEbiosystems Japan Co., Ltd.) or any common techniques in syntheticorganic chemistry. When necessary, the crude peptide products obtainedby these techniques may be purified by common purification techniquessuch as reversed-phase high-performance liquid chromatography andcrystallization.

The amino acid sequence of the precursor polypeptide of the peptide, aswell as the base sequence encoding the precursor polypeptide inaccordance with the present invention, may be determined in thefollowing manner:

Using a primer synthesized based on the amino acid sequence of thepresent peptide obtained in the above-described manner, total RNAprepared from the brains of O. vulgaris is subjected to the reversetranscriptase polymerase chain reaction (RT-PCR) to determine the genesequence of about 750 bp. Subsequently, the 5′-RACE and the 3′-RACEtechniques are used to determine the gene sequence on the 5′-end and the3′-end, respectively. According to the present invention, it wasdetermined that the precursor polypeptide of the present peptide has anamino acid sequence shown as SEQ ID NO: 2 and the gene encoding theprecursor polypeptide has a base sequence shown as SEQ ID NO: 3.

Accordingly, the precursor polypeptide of the peptide of the presentinvention, as well as the peptide of the present invention, can beproduced using recombinant DNA techniques. When it is desired to producethe pre-cursor polypeptide by using a recombinant DNA technique, avector incorporating for example the gene of SEQ ID NO: 3 may beintroduced into hosts for transformation. The hosts are subsequentlycultured or grown, and the precursor polypeptide of interest is isolatedand purified from the hosts or the culture solution of the hosts.

To obtain the peptide of interest from the precursor polypeptide, theprecursor polypeptide may be processed and modified using an enzyme. Ifnecessary, the peptide product may be subjected to an isolation or apurification process.

The peptides of the present invention each have an activity to enhancethe heart beat of O. vulgaris and have a structure quite analogous toGnRH (peptide) of vertebrates. The peptides also act on the pituitarygland of quails to cause the release of luteinizing hormone.Furthermore, studies of the peptides of the present invention suggestedthat bird GnRH is related to the activity to enhance the heart beat inoctopuses. These observations collectively indicate that the peptide inaccordance with the present invention is in fact a hormone (peptide)involved in the release of gonadotropins in octopuses and otherinvertebrates, as well as in vertebrates.

Accordingly, the peptide of the present invention can serve as a usefulmaterial both for studying biology of octopuses including mechanisms oftheir reproduction and for studying structural correlations betweenGnRHs of invertebrates and those of vertebrates. Thus, the peptide willbe of significant usefulness in the study of animal evolution. Also, thepeptide of the present invention is expected to find use not only in thedevelopment of new substances with the activity ofgonadotropin-releasing hormone, but also in the development of newmedicines and pesticides using the peptide itself or peptide analoguesthereof, as well as of new techniques for the production of juvenileoctopuses.

For example, the peptide of the present invention may be used as anactive ingredient in a medicine and may be formulated along with apharmacologically acceptable excipient into proper oral or parenteralpreparations, such as capsules, tablets, and injections. Specifically,the peptide of the present invention may be admixed with an excipient,such as lactose, starch or a derivative thereof, or a derivative ofcellulose, and is encapsulated in gelatin capsules to form a capsulepreparation.

When it is desired to form the peptide of the present invention into theform of tablets, a binder, such as carboxymethylcellulose sodium,alginic acid, and gum arabic, and water are added to the peptide inaddition to the aforementioned excipient. The mixture is then kneadedand, if necessary, is formed into the form of granules. Subsequently, alubricant, such as talc and magnesium stearate, is added to the mixtureand the mixture is formed into tablets by a common compressiontablet-making machine.

When it is desired to form the peptide of the present invention intopreparations for parenteral administration, the peptide may bedissolved, along with a solubilizer, into sterilized distilled water orsterilized saline and is placed in sealed ampoules to provide apreparation for injection. When necessary, a stabilizer, a bufferingagent, or other additives may be added to the preparation.Alternatively, the peptide of the present invention may be placed invials in the form of powder so that, upon use, the powder can bedissolved in sterilized distilled water to form a solution preparation.These parenteral preparations may be delivered by intravenousadministration or intravenous drip infusion.

Preferably, the dosage of the preparation containing the peptide of thepresent invention as an active ingredient is adjusted in considerationof various factors, including the disease to be treated, symptoms of thepatient, severity of the symptom, age of the patient, and route ofadministration. In case of oral administration, the preparation istypically administered at a dose of 0.1 to 1,000 mg/day/human,preferably at a dose of 1 to 500 mg/day/human, whereas in case ofparenteral administration, the preparation may be administered at a dosethat is about 1/100 to about ½ the dose for oral administration.

Because of its nature as GnRH, the peptide of the present invention canbe administered to cephalopods such as octopuses to cause the animals toreach sexual maturation at an earlier stage, so that the animals can beinduced to start laying eggs at an earlier stage. Breeding and culturingthese animals can facilitate production of juvenile cephalopods.

EXAMPLES

The present invention will now be described in detail in the followingwith reference to Examples, which are not intended to limit the scope ofthe invention in any way.

Example 1 Isolation of Cardioactive Peptides from O. vulgaris (a) CrudeExtraction

Brain tissue (including optic glands, optic tracts, and optic lobes) wastaken from 100 specimens of Octopus vulgaris and was frozen in liquidnitrogen. The frozen tissue (180 g) was boiled in 900 ml distilled waterfor 10 min. After cooling, acetic acid was added to a concentration of3% and the tissue was homogenized and then centrifuged at 4° C. at12,000×g for 30 min to obtain a supernatant. To the resulting pellet,200 ml of 3% acetic acid was added and the mixture was homogenized againfor extraction. Subsequently, the mixture was centrifuged under the sameconditions to obtain a supernatant. The supernatant was collected andconcentrated in vacuo to a volume of about 200 ml to give crudeextracts.

(b) Adsorption to C18 Cartridge

To the crude extracts obtained in the step (a) above, 5 ml of 6 M HClwas added and the mixture was centrifuged at 4° C. at 12,000×g for 30nm. The resulting supernatant was passed through Sep-Pak® Vac C18cartridge (10 g, 35 cc, manufactured by WATERS Co., Ltd.) to allow thesolutes to be adsorbed onto the cartridge. The cartridge was then washedwith 200 ml of 0.1% trifluoroacetic acid (TFA hereinafter), and theretained materials were then eluted with 100 ml of 60% methanol/0.1%TFA. The eluate was concentrated in vacuo and was freeze-dried to obtain150 mg dried products.

(c) Reversed-Phase High-Performance Liquid Column Chromatography (1)

Using Capcell pak C18 UG80 (5 μm, 10 mm×250 mm, manufactured by SHISEIDOCo., Ltd.), the dried products obtained in the step (b) above weresubjected to the reversed-phase high-performance liquid columnchromatography (reversed-phase HPLC). Specifically, the dried productsin 0.1% TFA (pH 2.2) were passed through the column at a flow rate of1.5 ml/min and the retained materials were eluted with a linear gradientof acetonitrile in which the concentration of acetonitrile in TFA waslinearly varied from 0% to 60% over 60 min. The eluate was collected in3 ml fractions based on the UV-absorbance at 215 nm.

Each fraction was bioassayed according to the scheme described inExample 5 below and, as a result, it was found that a fraction elutedwhile the concentration of acetonitrile was varied from 26% to 30% hadan activity to enhance the heart beat of O. vulgaris.

(d) Cation-Exchange Column Chromatography

Using TSK gel SP-5PW (10 μm, 7.5 mm×75 mm, manufactured by TOSOH Co.,Ltd.), the active fraction obtained in the step (c) above was subjectedto the cation-exchange column chromatography (cation-exchange HPLC).Specifically, the fraction was loaded onto the column and was elutedwith a 10 mM phosphate buffer (pH 7.0) at a flow rate of 1.0 ml/minwhile the concentration of NaCl in the phosphate buffer was linearlyvaried from 0 M to 0.6 M over 60 min (linear gradient). The eluate wascollected in 2 ml fractions and each fraction was bioassayed. Theactivity was found in the fraction eluted when the concentration of NaClwas 0 M.

(e) Reversed-Phase HPLC (2)

Using Capcell pak C18 UG80 (5 μm, 4.6 mm×150 mm, manufactured bySHISEIDO Co., Ltd.), the active fraction obtained in the step (d) abovewas subjected to the reversed-phase HPLC. Specifically, the fraction wasloaded onto the column and was eluted with 0.1% TFA (pH 2.2) at a flowrate of 1.0 ml/min while the concentration of acetonitrile in TFA waslinearly varied from 10 to 30% (linear gradient). The eluate wascollected in 1 ml fractions. The activity was found in the fractioneluted when the concentration of acetonitrile was about 19.5%.

(f) Reversed-Phase HPLC (3)

Using Capcell pak C18 UG80 (5 μm, 4.6 mm×150 mm, manufactured bySHISEIDO Co., Ltd.), the active fraction obtained in the step (e) abovewas subjected to the reversed-phase HPLC. Specifically, the fraction wasloaded onto the column and was eluted with 0.1% TFA (pH 2.2) having anacetonitrile concentration of 18% at a flow rate of 0.5 ml/min. Acompound that exhibited a single peak was obtained at a retention timeof 17.5 min.

The compound was designated as Compound (1). A diagram showing theresults of the reversed-phase HPLC was shown in FIG. 1.

Example 2 Structure Determination of Compound (1)

The structure analysis of Compound (1), purified in Example 1, byShimadzu PSQ-1 protein sequencer (manufactured by SHIMADZU Co., Ltd.)could not determine the amino acid sequence of the peptide due to themodified N-terminal. Thus, the amino acid composition of the peptide wasanalyzed instead. The results are shown in Table 1 below.

TABLE 1 Amino acid composition of the peptide Amino acid pmol Molarratio Asx 51.1 1.5 Glx 42.6 1.3 Ser 32.7 1.0 Gly 70.0 2.1 His 57.9 1.7Pro 37.1 1.1 Tyr 24.2 0.7 Phe 33.8 1.0

The molecular weight of Compound (1) was determined by Q-TOF (Micromass,manufactured by UK Co., Ltd.). The measurements are shown in Table 2below.

TABLE 2 Mass spectrum data of the peptide Molecular Calculated valueCalculated value Compound formula (M + 2H)²⁺ (M + 2H)²⁺ Before treatmentC₆₆H₈₀N₂₀O₁₇ 713.30 713.30 with enzyme After treatment C₆₁H₇₅N₁₉O₁₅657.79 657.85 with enzyme

It was determined from the analysis of the amino acid composition andthe measurement of the molecular weight that the N-terminal of Compound(1) includes a pyroglutamic acid residue. Accordingly, Compound (1) wastreated with an enzyme capable of cleaving off the first pyroglutamicacid residue (pyroglutamate aminopeptidase, manufactured by TAKARA SHUZOCo., Ltd.) and was again analyzed by the protein sequencer.

Consequently, the amino acid sequence of Compound (1) was determined,starting from the second amino acid residue, and the presence of Trp,which cannot be detected by the composition analysis, was verified.

The amino acid sequence determined is shown in Table 3 below.

TABLE 3 Amino acid sequence of the peptide after treatment with enzyme(pmol) Cycle Amino acid 1 Asn 27 2 Tyr 25 3 His 7 4 Phe 23 5 Ser 3 6 Asn16 7 Gly 12 8 Trp 3 9 His 2 10 Pro 5 11 Gly 4

To make sure that the enzyme treatment had worked as intended, themolecular weight of Compound (1) after the treatment was againdetermined by Q-TOF (Micromass, manufactured by UK Co., Ltd.) in thesame manner and, as a result, it was verified that the firstpyroglutamic acid residue had been cleaved off. The results are alsoshown in Table 2 above.

Through the foregoing instrumental analyses, it was determined thatCompound (1), a neuropeptide isolated and purified from O. vulgaris, hasthe following amino acid sequence (1):

(1) (SEQ ID NO: 1) <Glu-Asn-Tyr-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly- NH₂wherein <Glu represents pyroglutamic acid.

Example 3 Synthesis of Compound (1) by the Solid Phase Method

Using a fully automated peptide synthesizer 433A (PE Biosystems Japan),Compound (1) was synthesized according to FastMoc® chemistry.

In the synthesis, an Fmoc-NH-SAL-A resin (manufactured by WATANABECHEMICAL INDUSTRIES, Ltd.) to serve as a solid support was used, alongwith pyroglutamic acid, Fmoc-Asn-(Trt), Fmoc-Tyr-(tBu), Fmoc-His-(Trt),Fmoc-Phe, Fmoc-Ser-(tBu), Fmoc-Gly, Fmoc-Trp-(tBoc), and Fmoc-Pro.

As used herein, Fmoc represents 9-fluorenylmethoxycarbonyl, tBurepresents t-butyl, Trt represents trityl, and tBoc representst-butyloxycarbonyl.

A 10 ml mixture of 2.5% 1,2-ethanedithiol/2.5% water/95% TFA containing100 mg 2-methylindole was used for the purposes of cleavage of the crudepeptide from the peptide resin upon completion of the reaction anddeprotection of the crude peptide. After the reaction, the reactionmixture was filtered. Ether was then added to the filtrates to cause thepeptide to precipitate. The precipitates were washed three times withether to obtain about 100 mg of crude peptide, about 10 mg of which wasthen purified by the reversed-phased HPLC to obtain about 6 mg ofpurified peptide.

When subjected to the reversed-phase HPLC using Capcell pak C18, thepurified peptide eluted exactly at the same retention time as Compound(1). Also, the synthetic peptide exhibited the same cardioactivitytoward the heart beat of O. vulgaris as did the naturally-occurringpeptide.

Example 4 Synthesis of Compounds (2) and (3) by the Solid Phase Method

Peptides represented by the following amino acid sequences (2) and (3)were synthesized in the same manner as in Example 3 above:

(2) (SEQ ID NO: 5) <Glu-Asn-Tyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- NH₂(3) (SEQ ID NO: 6) <Glu-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH₂wherein <Glu represents pyroglutamic acid.

Example 5 Measurement of the Heart Beat Activity of O. vulgaris

The heart beat activity of O. vulgaris was measured according to themethod proposed by Morishita, et al. (Fumihiro Morishita, Biochem.Biophys. Res. Commun., 240, 354-358 (1997)). Specifically, the heart wastaken from O. vulgaris and the atria were removed from the heart. Acannula was inserted from each atrium into the ventricle and was at thesame time connected to a chamber (80 ml in volume) by tying with acotton thread. The aorta was pinched to prevent leakage of liquid fromthe ventricle and was connected to a tension transducer. This completedpreparation of the heart to serve as a specimen for the assay. Eachcannula was adjusted to permit a 1 to 2 ml/min flow of artificial seawater (containing 0.1% glucose). Each of the analytes to be assayed wasdissolved in 1 ml of the same artificial sea-water, and the resultingsolution was injected into the heart through the cannulae. The variationof the heart beat was recorded.

As test compounds, Compound (1) isolated from the brains of O. vulgaris,Compound (2), and cGnRH-II, a vertebrate GnRH isolated from chickens andused to derive Compound (2) therefrom, were used.

As shown in FIG. 2, the results indicate that Compound (1) enhanced theheart beat of O. vulgaris.

As shown in FIG. 3( b), Compound (2) also enhanced the heart beat of O.vulgaris by increasing both the heart rate and the amplitude of theheart beat. In contrast, cGnRH-II, as can be seen from FIG. 3( a), didnot have any effect on the heart rate or the amplitude of the heart beatof O. vulgaris.

Example 6 Effects on the Pituitary Glands of Quails

Cells taken from the pituitary glands of quails were inoculated onto a24-well plate at 7.5×10⁵ cells/well (substantially equivalent to twoanimals). The cells were then incubated for 3 days in a culture mediumcontaining fetal calf serum. The cells were incubated for another 24 hrsin 1 ml serum-free medium. After the incubation period, the peptidesolution was added to the culture medium to final concentrations of 0,10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ and 10⁻⁵ M, respectively, and the cultures werefurther incubated for 4 hrs. The supernatant was then collected fromeach culture. For each concentration, 5 samples were subjected toradioimmunoassay and measurements were taken. The data werestatistically analyzed by one-way analysis of variance (ANOVA).

The results are shown in FIG. 4. As shown, the statistical analysisrevealed that Compound (1), the peptide of the present invention,significantly increased the release of luteinizing hormone (LH).

When tested in the same manner, Compound (3) also proved to increase therelease of luteinizing hormone (LH).

Example 7 Determination of the Entire Amino Acid Structure of thePrecursor Polypeptide and Determination of the Base Sequence of the GeneEncoding the Polypeptide

(1) Preparation of Total RNA of the Brain of O. vulgaris

About 1 g of the brain of O. vulgaris was crushed in liquid nitrogen,dissolved in 10 ml TRIzol® reagent (GIBCO BRL Co., Ltd.), andhomogenized. At room temperature, the homogenates were allowed to standfor 5 min and were divided into 1 ml aliquots. To each aliquot, 200 μlchloroform was added and the mixture was stirred. The mixture was thencentrifuged (15,000 rpm, 15 min, 4° C.) in a cold centrifuge (SAKUMASEISAKUSHO Co., Ltd.). Following the centrifugation, the top aqueouslayer was removed and 0.5 ml isopropanol was added to the aqueous phase.The mixture was then allowed to stand for 10 min at room temperature andwas further centrifuged (15,000 rpm, 10 min, 4° C.) in the coldcentrifuge. The supernatant was removed and the pellet was resuspendedin 1 ml 75% ethanol. The suspension was centrifuged again (10,000 rpm, 5min, 4° C.). Subsequently, the supernatant was removed and the pelletwas air-dried for about 10 min. The RNA pellet was then dissolved in 101DPEC-treated water by incubating at 60° C. for 10 min. In this manner,about 3 mg of total RNA, was obtained.

(2) Degenerate 3′-RACE

The following degenerate primers were designed based on the amino acidsequence of the peptide isolated from the brain of O. vulgaris andsynthesized by an ordinary method:

Primer-1: (SEQ ID NO: 10)5′-CA(A/G)AA(C/T)TA(C/T)CA(C/T)TT(C/T)IIIAA(C/T) GG-3′ Primer-2: (SEQ IDNO: 11) 5′-(T/A) (C/G)IAA(C/T)GGITGGCA(C/T)CCIGG-3′wherein each letter is based on the definition in “Table of NucleotideCodes” (Biology experiment illustrated, supplement to Cell Engineering,SHUJUNSHA Co., Ltd. The same codes are used hereinafter.).

Using a 5′/3′-RACE Kit (Boehringer Mannheim Co., Ltd.), degenerate3′-RACE was performed according to the following protocol: 2 μg totalRNA, 4 μl cDNA synthesis buffer, 2 μl dNTP mix, 1 μl oligo dT-anchorprimer (12.5 pmol/μl), 1 μl AMV reverse transcriptase (20 units/μl), andDEPC-treated water were added to one another to give a total volume of20 μl. The mixture was incubated at 55° C. for 60 min and then at 65° C.for 10 min to synthesize 1st-strand cDNA.

Next, 1st-3′-RACE was performed according to the following procedure: 3μl 1st-strand cDNA, 5 μl 10×PCR buffer, 8 μL dNTP mix, 5 μl Primer-1(100 pmol/μl), 1 μl PCR anchor primer (12.5 pmol/μl), 0.5 μl TaKaRa ExTaq® (TAKARA SHUZO Co., Ltd.), and water were mixed with each other togive a total volume of 50 μl. The mixture was incubated at 94° C. for 5min, followed by 30 cycles of 94° C. for 30 sec, 49° C. for 30 sec, and72° C. for 2 min, which was followed by incubation at 72° C. for 5 min.GeneAmp PCR System 2400 thermal cycler (Perkin Elmer Co., Ltd.) was usedto carry out the PCR.

Subsequently, the 1st-PCR products were purified through a spin column(MicroSpin® S-400, manufactured by Amersham Pharmacia Co., Ltd.) andwere subjected to 2nd-3′-RACE according to the following procedure: 5 μl1st-PCR products, 5 μl 10×PCR buffer, 8 μl dNTP mix, 5 μl Primer-2 (100pmol/μl), 1 μl PCR anchor primer (12.5 pmol/μl), 0.5 μl TaKaRa Ex Taq®(TAKARA SHUZO Co., Ltd.), and water were mixed with each other to give atotal volume of 50 μl. The mixture was incubated at 94° C. for 5 min,followed by 30 cycles of 94° C. for 30 sec, 51° C. for 30 sec, and 72°C. for 1 min, which was followed by incubation at 72° C. for 7 min.

Five microliter of the reaction mixture was then electrophoresed on a1.5% agarose gel. Bands were observed at about 400 bp and about 600 bp,indicating amplification of two PCR products.

(3) Collection of PCR products

Five microliter of the PCR products was electrophoresed on a 1.5%agarose gel and the two PCR product bands formed at about 400 bp andabout 600 bp were cut out. Using Gel Extraction Kit (QIAGEN Co., Ltd.),DNA was collected from the gel. To confirm the results, 3 μl of each ofthe collected DNA fragments was electrophoresed on a 1.5% agarose gel.As a result, the two PCR products were observed at about 400 bp andabout 600 bp, respectively.

(4) Ligation of the PCR Products

The two PCR products, sized about 400 bp and about 600 bp, respectively,were individually purified through a spin column. For each of the PCRproducts, 3 μl of PCR product, 2 μl TA cloning vector pCR2.1(Invitrogene Co., Ltd.), and 5 μl Ligation High (TOYOBO Co., Ltd.) weremixed with each other, and the mixture was allowed to react for 1 hr at16° C. (ligation).

(5) Transformation of E. coli

Each of the two 10 μl ligation mixtures obtained in the step (4) abovewas mixed with highly competent E. coli DH5α (TOYOBO Co., Ltd.). Themixture was placed in an ice bath for 30 min and was then subjected toheat-shock at 42° C. for 50 sec, followed by chilling on ice for 2 min.After chilling, 1 ml SOC medium was added and the mixture was incubatedat 37° C. for 10 min. Subsequently, 35 μl X-gal was evenly spread ontothe surface of LB/Amp agar medium (LB containing 50 μg/ml ampicilin),and 50 μtransformant was inoculated onto the medium. The remainingtransformant was centrifuged at 10,000 rpm for 1 min to a volume ofabout 100 μl and the whole transformant was also inoculated onto theLB/Amp agar medium. The culture medium was incubated overnight at 37° C.

(6) Colony PCR

Using the colonies obtained above as a template, the colony PCR wasperformed. Specifically, the E. coli cells, 5 μl 10× reaction buffer, 5μl 2 mM dNTPs, 3 μl 25 mM MgCl₂, 0.5 μl M13FW primer (100 pmol/μl), 0.5μl M13RV primer (100 pmol/μl), 0.5 μl rtaq DNA polymerase (TOYOBO Co.,Ltd.), and water were mixed with each other to give a total volume of 50μl. The mixture was incubated at 90° C. for 10 min, followed by 30cycles of 94° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 1 nm.Three microliter of the reaction mixture was electrophoresed on a 1.5%agarose gel.

The M13FW and M13RV primers, the base sequences of which are shownbelow, were synthesized by an ordinary method:

M13FW: 5′-GTAAAACGACGGCCAGTG-3′ (SEQ ID NO: 12) M13RV:5′-GGAAACAGCTATGACCATG-3′. (SEQ ID NO: 13)

(7) DNA Sequence

The colony PCR products of the size of interest (about 600 bp and about800 bp) were purified through a spin column and were sequenced by DNASequencing Kit (PE Biosystems Co., Ltd.). ABI PRISM 310 Genetic Analyzer(PE Biosystems Co., Ltd.) was used for sequencing.

Through the sequence analysis by a gene-analyzing software GENETYX-MAC(SOFTWARE KAIHATSU Co., Ltd.), the cDNA sequence of the about 800 bpfragment was determined. A further DNA sequencing revealed that the cDNAsequence of the fragment of about 600 bp was in fact part of the cDNAsequence of the fragment of about 800 bp.

(8) 5′-RACE

The following primers were synthesized according to the base sequencesof the cDNA fragments.

5′-RACE-1: 5′-GGTCTGCAATCTCATTGACC-3′ (SEQ ID NO: 14) 5′-RACE-2:5′-TGCCAGTCAGGATTTCTCTC-3′ (SEQ ID NO: 15) 5′-RACE-3:5′-TCTTGGTGTTTGTCGGAAGG-3′. (SEQ ID NO: 16)

Using 5′/3′-RACE Kit (Boehringer Mannheim Co., Ltd.), 5′-RACE wasperformed according to the following protocol: 2 μg total RNA, 1 μl5′-RACE-1 (12.5 pmol/μl), 4 μl cDNA synthesis buffer, 2 μl dNTP mix, 1μl AMV reverse transcriptase (20 units/μl), and DEPC-treated water weremixed with each other to give a total volume of 20 μl. The mixture wasincubated at 55° C. for 60 min and then at 65° C. for another 10 min tosynthesize 1st-strand cDNA.

After purification through a spin column, the 1st-strand cDNA was mixedwith 2.5 μl reaction buffer and 2.5 μl 2 mM dNTP, and the mixture wasincubated at 94° C. for 3 min, followed by chilling on ice.Subsequently, 1 μl terminal transferase (10 units/μl) was added to themixture, and the mixture was incubated at 37° C. for 20 min and then at70° C. for another 10 min to synthesize dA-tailed cDNA.

Next, 1st-PCR and 2nd-PCR were conducted under the following conditions:

(a) 1st-PCR

Three microliter d-tailed cDNA, 5 μl 10×PCR buffer, 8 μl dNTP mix, 1 μl5′-RACE-2 (12.5 pmol/μl), 1 μl oligo dT-anchor primer (12.5 pmol/μl),0.5 μl TaKaRa Ex Taq® (TAKARA SHUZO Co., Ltd.) and water were mixed witheach other to give a total volume of 50 μl. The mixture was incubated at94° C. for 5 min, followed by 30 cycles of 94° C. for 30 sec, 55° C. for30 sec, and 72° C. for 3 min, which was followed by incubation at 72° C.for 5 min.

(b) 2nd-PCR

Five microliter of the 1st-PCR product purified through a spin column, 5μl 10×PCR buffer, 8 μl dNTP mix, 1 μl 5′-RACE-3 (12.5 pmol/μl), 1 μlPCR-anchor primer (12.5 pmol/μl), 0.5 μl TaKaRa Ex Taq® (TAKARA SHUZOCo., Ltd.) and water were mixed with each other to give a total volumeof 50 μl. PCR was then carried out by using the same cycle conditions asin the 1st-PCR.

The resulting 2nd-PCR product was electrophoresed on a 1.5% agarose gel.As a result, a band was observed at about 600 bp.

The base sequence of the 2nd-PCR product was determined according to theprotocol described in (4), (5), (6) and (7) above.

Through the foregoing analyses, the size of cDNA (about 700 bp) and thebase sequence were determined for the gene encoding the precursorpolypeptide of the peptide of the present invention. Also, the estimatednumber of amino acids (89 amino acid residues), as well as the aminoacid sequence, was determined for the precursor polypeptide of thepeptide of the present invention.

The amino acid sequence of the precursor polypeptide and the basesequence of the gene (cDNA) encoding the precursor polypeptide are shownas SEQ ID NO: 2 and SEQ ID NO: 3, respectively. Also, the part of theamino acid sequence of the precursor polypeptide that corresponds to thepeptide of the present invention is shown in FIG. 6.

In the figure, the part corresponding to the amino acid sequence of thepeptide of the present invention is underlined.

Example 8 Exemplary Preparation Tablets

Composition: Compound (1) 10 g lactose 125 g microcrystalline cellulose25 g corn starch 25 g 5% hydroxypropyl methylcellulose 100 ml magnesiumstearate 5 g

According to a known method, the above components were kneaded,granulated, dried and shaped into tablets to obtain tablets, eachweighing 190 mg and containing 10 mg of Compound (1) as an activeingredient.

INDUSTRIAL APPLICABILITY

As shown in FIG. 5, the peptide of the present invention, which acts toenhance the heart beat of O. vulgaris, has a structure highly analogousto gonadotropin-releasing hormone (peptide) of vertebrates. Togetherwith this finding, the fact that the peptide acts on the pituitary glandof quails to cause the release of luteinizing hormone implies that thepeptide is in fact a hormone (peptide) involved in the release ofgonadotropin in octopuses.

Thus, studying the activities of the present peptide will helpunderstand biology of octopuses including their breeding mechanisms, aswell as processes of animal evolution.

Not only does the peptide of the present invention serve as a usefultool in developing new substances with the activity ofgonadotropin-releasing hormone, but it also may lead to development ofnew medicines and pesticides that make use of the peptide itself orpeptide analogues, as well as of new techniques for the production ofjuvenile octopuses.

1. A peptide having the activity of gonadotropin-releasing hormone(GnRH) and having an amino acid sequence obtained by deleting aminoacids 2 and 3 from the N-terminal region of the peptide having thefollowing amino acid sequence (I): (I) (SEQ ID NO: 7)<Glu-Zaa-Zaa-His-Zaa-Ser-Zaa-Zaa-Zaa-Zaa-Pro-Gly- NH₂

wherein <Glu represents pyroglutamic acid and Zaa represents any aminoacid.
 2. The peptide according to claim 1 having the following aminoacid sequence (3): (3) (SEQ ID NO: 6)<Glu-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH₂

wherein <Glu represents pyroglutamic acid.
 3. An isolated precursorpolypeptide of a peptide having the activity of gonadotropin-releasinghormone (GnRH) and having an amino acid of SEQ ID NO:
 2. 4. A DNAsegment encoding the precursor polypeptide of claim 3, which is aprecursor of the peptide having the activity of gonadotropin-releasinghormone (GnRH).
 5. A DNA segment encoding a polypeptide having an aminoacid sequence represented by SEQ ID NO: 1 or SEQ ID NO:
 2. 6. A DNAsegment having a base sequence represented by SEQ ID NO:
 3. 7. A DNAsegment capable of hybridizing with the DNA segment of any of claims 4to
 6. 8. A DNA segment having a homology of 60% or higher to the DNAsegment of any one of claims 4 to
 6. 9. A vector incorporating the DNAsegment of any one of claims 4 to
 6. 10. A host transformed by thevector of claim
 9. 11. A method for producing a precursor polypeptide ofa peptide having the activity of gonadotropin-releasing hormone (GnRH),comprising the steps of: culturing or growing the host of claim 10; andcollecting the precursor polypeptide of the peptide having the GnRHactivity from the host or the culture solution of the host.
 12. Aprecursor polypeptide of a peptide having the activity ofgonadotropin-releasing hormone (GnRH), obtained by the method of claim11.
 13. A method for producing a peptide having the activity ofgonadotropin-releasing hormone (GnRH), comprising the steps of: excisinga precursor of the peptide having the GnRH activity from the precursorpolypeptide of the peptide having the GnRH activity, the precursorpolypeptide being obtained by the method of claim 11; and subsequentlymodifying the C-terminal and the N-terminal of the precursor.
 14. A DNAsegment having a homology of 60% or higher to the DNA segment of claim7.
 15. A vector incorporating the DNA segment of claim
 7. 16. A vectorincorporating the DNA segment of claim 8.